Pharmaceutical composition containing a safe extracts of fruits and vegetables for the treating and preventing of diabetes

ABSTRACT

The new pharmaceutical composition and processes are provided for treating and preventing diabetes. 
     The pharmaceutical composition is composed of three ingredients: Oleanolic acid, Saponins of Litchi and Kuguasu. 
     The pharmaceutical composition is nontoxic.

This application is a continuation of application Ser. No. 063,978,filed 6/18/87, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates in general to the treatment of diabetes. Moreparticularly, it relates to the method of reducing blood glucose andrepairing disorderly metabolism in the treatment of diabetes byadministering an effective amount of a pharmaceutical composition.

DESCRIPTION OF THE PRIOR ART

The major characteristic of diabetes is the body's inability to regulatethe level of glucose in the blood. Therefore, the goal of treatingdiabetes is reducing the blood glucose. In clinic, insulin and some oralhypoglycemic drugs, which include tolbutamide, tolazamide,acetohexamide, chlorpropamide, glyburide and glipizide, are availablefor treating diabetes. However, all mentioned above drugs have severaldisadvantages in therapeutic use. For example, some drugs are increasedthe risk of acute cardiovascular disease. All drugs are not effective intreating the following symptom: diabetic acidosis or in stressfulsituation such as infection and the degenerative diseases which cause bydiabetes. More particularly, as mentioned earlier drugs are noteffective in treating atheroschlerosis, lose the sight, maimed and deathbrought about by progressive vascular injury, and in fact, above diseaseis main lethal reason of diabetes.

Additional, it has been established in the prior art the oleanolic acidcan inhibited carrageenin-induced swelling in rats and inhibitedcapillary permeability in mice. Oleanolic acid also is used in the fieldof cosmetics.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide pharmaceuticalcomposition which is safety and high effective treating and preventingdiabetes disease.

In keeping with these objects and other objects which will becomeapparent hereinafter, the present invention resides, briefly state incomposition comprising a mixture of the following active ingredients:

1. Oleanolic acid extracted from fruit of Diospyros kaki L. F.;

2. Saponins of Litchi extracted from fruit or seed of Litchi chinensisSonn;

3. Kuguasu extracted from fruit of Momordica charantia L.

Above plants are fruit or vegetable which come into the fruit orvegetable market of U.S.

For the sake of convenience, composition comprising mixtures of theabove extracts will hereinafter be referred to as "Composition".

DETAILED DESCRIPTION

According to the present invention, it has been found the pharmaceuticalcomposition of these ingredients have a significant reducing bloodglucose and repairing disorderly metabolism at same time and to theiruse as medicine, particularly in the therapy of diabetes.

Diabetes is a disease that affects at least five percent of Americanpopulation. It is the third leading cause of death in the United States.The annual incidence of diabetes is 612,000. The economic impact ofdiabetes is enormous, estimated at 8 billion dollars one year. Nearly 10percent of working persons age 45 or older are diabetics. [Entmacher, P.S. (1983): in Diabetes mellitus, theory and practise, Edited by M.Ellenbeng and M. Rifkin, pp. 1053-1061. Medical Examination PublishingCo., New York.] It is the most common serious metabolic disorder.

Diabetes is a state of absolute or relative lack of functional insulin.It is not a single disease in the classic sense; but rather a clinicalsyndrome applied to a number of Pathogenetically heterogenous disorders.To be exact, Diabetes is disease characterized by abnormalities of theendocrine secretions of the pancreas resulting in disordered metabolismof carbohydrate, fat and protein, and in time, structural and functionalabnormalities in a variety of tissues. It also has been established inthe prior art that metabolism of carbohydrate, fat, protein andhormones, et al regulated by liver. The liver plays a key role inregulation metabolism of carbohydrate (including glucose) and isimportant in many other bodily functions. It manufactures bloodcoagulants, stores vitamins and minerals, produces enzymes, cholesterol,proteins and neutralizes substances that would harm the body. The livercan construct the storage form of many energy sources, for example,glycogen and fats. The liver can also convert glucose to protein andfat, protein into glucose, and fat into protein or glucose. Obviously,the liver plays a key role in regulation metabolism of diabetes.

For reason given above, "composition" which can reducing blood glucoseand repairing disorderly metabolism including increasing synthesis ofRNA and protein in injured liver at same time, it is very important fortreating and preventing diabetes.

In addition, Composition is very safe because all plants are fruit orvegetable.

Composition can administered to patients in the form of capsulescontaining a powdered mixture of the active ingredients in appropriateproportions. Alternatively, tablets can be prepared comprising theactive ingredients and pharmaceutically acceptable binders, excipients,lubricants, sweeteners and coatings. A syrup or elixir may be preparedby dissolving Composition in alcohol or water together with suitablepreservatives, sweeteners, dyes and flavoring agents. Ampules or vialsfor injection may likewise be prepared, with the Composition as preparedfor oral administration being purified through further sterilization andthe addition thereto of distilled water and other suitable solvents andadditive known in the pharmaceutical art.

The Composition dosage units prepared according to the invention can beadministered to patients with a very safe and in reducing blood glucoseand repairing disorderly metabolism.

Oleanolic acid has the following structural formula: ##STR1##

The following specific examples will provide detailed illustrations ofmethods of producing Composition according to the present invention andpharmaceutical dosage units containing Composition. Moreover, exampleswill be given of pharmaceutical testing performed with Composition whichdemonstrates its effectiveness in treating and preventing diabetes.These examples are not intended, however, to limit or restrict the scopeof the invention in any way, and should not be construed as providingconditions, parameters, reagents, or starting materials which must beutilized exclusively in order to practice the present invention.

EXAMPLE 1 Extraction of Oleanolic acid

Oleanolic acid extracted from fruits or leaves of Diospyros kaki L.F.,or Ligustrum lucidum Ait. The fruits or leaves of Diospyros kaki L.F.;or Ligustrum lucidum Ait dried and powdered. 3 liter of 90% ethanol wasadded to 1 kg of dried powder and allowed to stand for one day at roomtemperature and then refluxed in a water bath for 6 hours. The refluxingwas repeated twice by collecting the ethanol, replacing it with an equalvolume of fresh 90% ethanol and refluxing for 6 hours. The refluxedethanol was cooled and filtered and the filtrate combined with theextract filtrate. Then recovered by reduce pressure distillation and theresidue dissolved in hot water. Solution of water was filtered and thefilter cake (1) saved. The filter cake (1) dissolved in 95% ethanol andthen PH adjusted to 11 with 1 N NaOH. Solution of 95% ethanol allowed tostand and filtered. Then PH adjusted to 1 with 1 N HCl. Stand. Filteredand the filter cake (2) saved. PH 11 solution of water was added tofilter cake (2) and then the solution was heated to boil. Filtered.Filter cake (3) saved. The filter cake (3) washed twice with water andthen dissolved in ethanol and then PH adjusted to 1 with 1 N HCl.Needles crystals are obtained. Wash with water. A melting point is about310° C.

EXAMPLE 2 Extraction of Saponine of Litchi

Saponins of Litchi extracted from fruit or seed of Litchi chinensisSonn. The seed or fruit of Litchi chinensis Sonn dried and powdered.2,000 ml of 95% ethanol was added to 1,000 g or dried powder and allowedto stand for one day at room temperature. The solution was filtered andthe extract filtrate saved. 2000 ml of ethanol was added to the residueand refluxed in a water bath for 6 hours. The refluxing was repeatedtwice by collecting the ethanol, replacing it with an equal volume offresh 95% ethanol and refluxing for 6 hours. The refluxed ethanol wascooled and filtered and the filtrate combined with the extract filtrate.Ethanol was then recovered by reduced pressure distillation and theresidue dissolved in 500 ml of distilled water. The lipid component wasremoved with 3 changes of ether by adding 500 ml to the water phase foreach extraction. An equal volume of water-saturated butanol was added tothe final water phase and the butanol was then distilled under reducedpressure. The residue powder was dissolved in 500 ml of ethanol and2,000 ml of acetone was added to the ethanol with constant stirringwhile a precipitate formed. The precipitate was washed twice each withacetone and ether and dried.

EXAMPLE 3 Extraction of Kuguasu

Kuguasu extracted from fruit of Momordica charantia L. The fruit ofMomordica charantia L. dried and powdered. 5 liter of 90% ethanol addedto 1 kg of dried powder and adjusted PH of solution to 2.5 with HCl.Stirred. Centrifugalized. Supernatant was saved. SnCl₂ (50%) was addedto supernatant and adjusted to PH to 6.8 with NH₄ OH. Centrifugalized.Precipitate was saved. The precipitate dissolved in acid solution.Solution of NaCl was added to the acid solution and precipitate wasformed. The precipitate wash with acetone and dry under vacuum.Powdered.

EXAMPLE 4 Preparation of fine composition

Fine composition according to the present invention consists of:

    ______________________________________                                                      Weight Percent                                                  ______________________________________                                        Oleanolic acid   5 to 30%                                                     Saponins of Litchi                                                                            10 to 50%                                                     Kuguasu         30 to 90%                                                     ______________________________________                                    

The dry ingredients or derivate of ingredients prepared in accordancewith the present invention, may be incorporated tablets, capsules,syrups or other form by conventional method.

The tablets can be prepared by mixing the Composition with binders,excipients, disintegration agents, lubricants and sweetness. Examples ofwidely used, pharmaceutically acceptable tablet ingredients are cornstarch or gelatin as binders, dicalcium phosphate as an excipients, cornstarch, potato starch or alginic acid and disintegration agents,magnesium stearate as a lubricant, and sucrose or lactose as sweeteningagents. The tablets may be coated with shellac or sugar to facilitateswallowing. The preferred weight ranges of the composition in tabletsare the same as given above for capsule dosage forms. The most preferredweight values for the components are, as in the case of capsules, 50-100mg of dry ingredients per dosage unit.

Elixirs or syrups may be prepared by dissolving Composition in water orethanol and, if desired, adding a sweetener such as sucrose, a suitablepreservative, a dye (chosen according to the desired coloration) and aflavoring agent, such as an orange or cherry flavor. The concentrationranges of the Composition per teaspoon (5 milliliters) of syrup are thesame as given above for capsule and tablet dosage units.

EXAMPLE 5 Preparation of crude composition

Crude Composition is extracted from as mentioned above plants by ethanoland water. Proportion of plants, for example, is as following (byweight):

    ______________________________________                                                        Weight Percent                                                ______________________________________                                        Diospyros kaki L.F.,                                                                             5 to 50%                                                   Litchi chinesis 10 to 70%                                                     Momordica charantia L.                                                                          10 to 70%                                                   ______________________________________                                    

The tissues of plants were dried and powdered. 5 liters distillatorywater was added 1 kg of dried powder. The solution was heated to boiland simmered for one hour after boiling. This water extraction wasrepeated two times. Combined and filtered. The filtrate was concentratedunder reduced pressure to approximately 500 ml. Then 2,000 ml of 90%ethanol was added to 500 ml water solution. Stir. Stilled. Filtered.Residue and filtrate (A) was obtained. 1,000 ml 90% ethanol was added toresidue. Stir. Stilled. Filtered. Filtrate (B) was obtained.

Combined filtrate (A) with (B). Then total filtrate was concentrated tosyrup under reduced pressure distillation. Ethanol was recovered. Syrupdried under vacuum drying. Granulated to final powder. Weight of everycapsule and table is about 200-500 mg. Crude-Composition is similar tofine-composition in pharmacological property.

The following examples are related to pharmacological tests.

EXAMPLE 6 Hypoglycemic effect of Composition

Experiments use alloxan diabetic mice. Male mice 18-22 g were used inthese experiments. The diabetic mice had high blood glucose, produced bya single dose of alloxan 75 mg/kg intravenously. Inject 2 ml of normalsaline into the peritoneal cavity of mouse for control and 100 mg/kgComposition group daily. Blood samples were collected from ocular venousplexus of mice.

The blood glucose levels were determined according to hexokinase method.The procedure is as the following:

A. Reagents

1. Vial B, containing NADP. Reconstitute by adding 15.5 ml water andgently swirling.

2. Vial A. Add the entire contents of vial B to vial A and dissolve bygently inversion. According to the manufacturer, the reagent has thefollowing composition:

a. Tris buffer, pH 7.5, 50 mmol/L

b. ATP, 0.5 mmol/L

c. NADP⁺, 0.45 mmol/L

d. Mg⁺⁺, 17 mmol/L

e. Hexokinase, 666 U/L

f. G6PD, 333 U/L

3. Stock Standard Glucose, 10.0 g/L. Dissolve 1.0 g pure anhydrousD-glucose in water containing 1.0 g benzoic acid solution.

4. Working Glucose Standards. Prepare standards of 50, 100, 200, and 400mg/dl by appropriate dilution of Stock Standard with benzoic acidsolution.

B. Procedure

1. Place 1.5 ml prepared reagent in a series of cuvets for standard,unknowns, and control serum, respectively.

2. Appropriate blanks are set up by placing 1.5 ml of 9 g/L NaCl in aseries of cuvets.

3. Add 10 μl of standard, unknowns and control serum to the appropriatecuvets [tests and blanks]. Cover with Parafilm and mix. Place in 37° C.incubator for a faster reaction. It is permissible to carry out thereaction at room temperature, even though it takes longer for thereaction to go to completion.

4. After incubating for 5 or 10 minutes, read the absorbance of eachcuvet at 340 nm and check again a few minutes later to insure that anend point has been reached.

5. Calculation. Let ΔA be the difference in absorbance reading betweeneach test and its blank. ##EQU1## Where C=concentration of standard inmg/dl. ΔAμand ΔA, are the respective differences in absorbance betweenunknown or Standard and its blank.

                  TABLE 1                                                         ______________________________________                                                       Control  Composition                                           ______________________________________                                        Blood glucose level (mg/dl)                                                                    245.3 ± 36.6                                                                          171.5 ± 18.9                                   Number of samples                                                                              20         20                                                P                <0.1                                                         ______________________________________                                    

From above results, it is apparent that Composition can obviouslydecrease blood glucose levels in diabetic model.

EXAMPLE 7 Effect of Composition on binding insulin receptor

Rats were sacrificed by a blow on the head, and their epididymal adiposetissue were quickly removed. The fat cell were isolated from the adiposeby the procedure of Rodbell (Rodbell, M.: J. Biol. Chem, 239:375, 1964).In dulbecco buffer PH 7.4 containing collagenase (3 mg/ml) and albumin(40 mg/ml).

¹²⁵ I-labeled insulin (¹²⁵ I-insulin) was at specific activities of100-200μ Ci/μg. IgG was prepared from heparinized plasma. The lgGfraction of serum from the patient with the highest concentration ofantireceptor antibody activity (B-2) was prepared from the ammoniumsulfate precipitate by ion exchange chromatography of DEAE-cellulose.Antireceptor antibodies were assayed by methods of inhibition of ¹²⁵I-insulin binding to cultured human lymphoblostoid cells. The cells wereprepared: 2-4×10⁶ cells/ml of adipocytes cells were washed three timesfor 10 minutes at 37° C. and nondissociable radioactivity was extractedin 1% triton X-100. ¹²⁵ I-insulin binding to isolated rat adiposyteswere performed at 37° C. in krebs-ringer bicarbonate medium (PH 7.4)containing bovine serum albumin and bacitracin (100 U per milliliter).After adipocytes had been incubated with ¹²⁵ I-insulin for 30 minutes at37° C., the cell were precipitated from the medium by centrifugation.The radioactivity in the pellet was counted.

                  TABLE 2                                                         ______________________________________                                                        Control                                                                              Composition                                            ______________________________________                                        The binding of .sup.125 I-insulin                                                                100%    120.0 ± 14.0%                                   receptor                                                                      The number of samples                                                                           10       10                                                 P                 <0.05                                                       ______________________________________                                    

From above results, it is apparent that Composition can obviouslystimulate binding insulin receptor with insulin.

EXAMPLE 8 The effect of Composition on synthesis of protein

The 20-22 g male mice were used in experiments. The mice were injectedwith CCl₄. The dosage of Composition was 75 mg/kg injectedintraperitoneally. The control mice were injected with same volume ofnormal saline. The mice were sacrificed by decapitation, their liver wasquickly excised and placed immediately in cold Medium which consistingof 0.25 M sucrose, 0.065 M potassium chloride, 0.035 M potassiumbicarbonate, 0.01 M magnesium chloride and 0.05 M tris (hydrodymethyl)aminomethane (Tris), adjusted to pH to 7.5 with HCl. The liver wascleaned of excess fat before the wet weight was measured. The liver fromeach animal were homogenized in each experiment. All operations wereperformed at 4° C. Each liver was homogenized in 10 ml of cold Medium.Using a Teflon and glass homogenizer immersed in ice. The homogenate wascentrifuged at 1000 g for 10 minutes to remove large cellular particles.The resulting supernatant fluid was filtered through four layers ofcloth to remove as much fatty material as possible. The filtrate wascentrifuged at 37,000 g for 30 minutes. The sediment was discarded, andthe resulting postmitochondrial fraction was used for the assay oftranslation. Protein concentration was measured by the biuret procedure(J. Biol Chem 177:751, 1949), using crystalline bovine serum albumin asa standard. The rate of translation was determined in an assay systemcontaining: 0.2 ml of 0.01 M ATP, 0.2 ml of 0.05 M phospho puruvate,0.05 ml of a ³ H-amino acid mixture (containing approximately 5×10⁶cpm), 0.05 ml of crystalline pysuvate kinase (1 mg/ml), 0.1 ml of waterand 1.0 ml of the postmitochondrial preparation in Medium in a totalvolume of 2 ml. The postmitochondrial preparation was added last toinitiate the reaction, and the mixture was incubated for 30 minutes at37° C. Under the conditions of the experiment, translation was astraight-line function of time for at least 45 minutes. The course ofthe reaction was halted by the addition of 5 ml of 10% trichloroaceticacid (TCA). Control tubes were prepared by adding all of the componentsof the reaction mixture into 5 ml of 10% TCA. The precipitated proteinswere collected on a 0.45-μm membrane filter, using vacuum filtration.The collected precipitate was washed two times with 20 ml portions of10% TCA and dried in an oven at 80° C. for 10 minutes. The dried filterswere placed in scintillation vials containing 20 ml of Aquasol, and theradioactivity which had been incorporated into protein was measured in aliquid scintillation counter.

                  TABLE 3                                                         ______________________________________                                                      Control                                                                              Composition                                              ______________________________________                                        CPM/mg Proteins 639 ± 81                                                                            891 ± 88                                          Number of sample                                                                              18       18                                                   P               <0.01                                                         ______________________________________                                    

EXAMPLE 9 The effects of Composition on ribonucleic acid (RNA)

The method of animal is like procedure of example 8. ³ H-uridine (10μCi/100 g body weight) was injected intraperitoneally into mice 20minutes prior to sacrifice. Their liver was quickly excised. Livers werewashed with cold 0.25 M sucrose containing 3.3 mM CaCl₂ and minced withscissors. The mince was then homogenized with 3 volumes of the samesolution in a Potter's homogenizer with a glass pestle and centrifugedat 1000×g for 10 minutes. The sediment was homogenized with 3 volumes of0.25 M sucrose-3.3 mM CaCl₂ in a Potter's homogenizer with a Teflonpestle. The homogenates were filtered through 4 layers of gauze. Eightvolumes of 2.2 M sucrose was added and the mixture was centrifuged at40000×g for 1 hour to sediment the nuclei. Purified nuclei were washedwith 0.6 N perchloric acid, ethanol and ether. To the residues was added0.5 N KOH and the mixture was incubated at 37° C. for 18 hours, followedby acidification to remove deoxyribonucleic acid (DNA) and proteins asprecipitates. After centrifugation the supernatant was neutralized withKOH. Radioactivity incorporated into nuclear RNA and the amount of RNAwere determined using aliquots of this supernatant. Radioactivity wascounted in a scintillation spectrometer with solution, the compositionof which was as follows: one liter of the solution contained 50 ml ofmethanol, 10 ml of ethyleneglycol, 60 g of naphthalene, 4 g of2,5-diphenyloxazole, 0.2 g of 1,4-bis [2(5 phenyloxaxolyl)]-benzene anddioxane.

                  TABLE 4                                                         ______________________________________                                                     Control    Composition                                           ______________________________________                                        Specific radioactivity                                                                       18090 ± 1819                                                                            21888 ± 2079                                   (CPM/mg RNA)   (n = 18)     (n = 18)                                          P              <0.01                                                          ______________________________________                                         n: the number of samples                                                 

EXAMPLE 10 Safety of Composition

1. The acute LD₅₀ of fine composition was found to be 1469 mg/kginjection in abodominal cavity in mice.

2. L.D₅₀ of crude-Composition: 5.4 g/kg injection in abodominal cavityin mice.

3. Each dose for an adult is 500-1000 mg. Using 50 kg as the averageweight of an adult the dosage is 1-10 mg/kg, therefore, it is very safe.

4. As to subacute toxicity tests, a dosage corresponding to 50 times theclinical dose is administered continually for two months, and noside-effects have been observed. The electro-cardiogram and functions ofliver and the kidney have not been affected and no injuries whateverhave been observed in the tissue slices of the heart, liver, spleen,lungs, kidneys and adrenal.

The preparation of Composition is simple and can be accomplished by theextraction methods set forth above or any conventional methods forextracting the active ingredients. The novelty of the present inventionresides in the mixture of the active ingredients in the specifiedproportions at invention and in the preparation of dosage units inpharmaceutically acceptable dosage form. The term "pharmaceuticallyacceptable dosage form" as used hereinabove includes any suitablevehicle for the administration of medications known in thepharmaceutical art, including, by way of example, tablets, capsules,syrups, elixirs, and solutions for parenteral injection with specifiedranges of Composition.

It will thus be shown that there are provided compositions and methodswhich achieve the various objects of the invention, and which are welladapted to meet the conditions of practical use.

As various possible embodiments might be made of the above invention,and as various changes might be made in the embodiments set forth above,it is to be understood that all matters herein described are to beinterpreted as illustrative and not in a limiting sense.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended claims:
 1. A pharmaceutical composition forthe treatment of diabetes comprising:oleanolic acid 5-30 wt. %; saponinsof litchi 10-50 wt. % and Kuguasu 30-90 wt. %.
 2. A dietetic compositionfor treatment of diabetes comprising ethanol extracts of dried andpowdered fruits, seeds and leaves of:Diospros kaki L.F. 5-50 wt. %Litchi chinesis Sonn 10-70 wt. % and Momordica charantia L. 10-70 wt. %.3. A method for treating a diabetic patient comprising administering tosaid patient an effective anti-diabetic dose of a composition comprisingoleanolic acid 5-30 wt. %; saponins of litchi 10-50 wt. % and Kuguasa30-90 wt. %.
 4. A method for treating a diabetic patient comprisingadministering to said patient an effective anti-diabetic dose of acomposition of claim 2.